Project summary/abstract Recent evidence indicates that epigenetics plays an essential role in modulating tightly-coordinated transcription factors involved in cartilage development. Epigenetic modifications such as acetylation/deacetylation or methylation/demethylation have been intensively investigated in the chondrogenesis of adult stem cells such as mesenchymal stem cells (MSCs). However, there is a significant gap in our understanding of how epigenetics regulates chondrocyte specification from pluripotent stem cells (PSCs) in vitro or during cartilage development in vivo. Thus, the long-term goal of our proposed work is to unravel novel epigenetic mechanisms governing chondrocyte specification and cartilage development at the cellular and molecular levels. In our recent work of identifying gene regulatory networks of chondrogenesis of human induced pluripotent stem cells (hiPSCs) via single-cell RNA-Seq (scRNA-Seq), we reveled that PRDM16, a histone methyltransferase, was significantly up- regulated at the stage of chondrocyte specification from mesodermal cells, and its expression level was positively correlated with several key chondrogenic transcription factors. These results imply that PRDM16 may play an essential role in regulation chondrocyte cell fate decision. Thus, we hypothesize that PRDM16 is a critical epigenetic regulator in chondrogenic lineage specification and cartilage development. To test this hypothesis, our approaches in the K99 phase are to: 1) Elucidate the functional role and gene regulatory networks of PRDM16 in hiPSC chondrogenesis in vitro by scRNA-Seq of the cells with overexpression or knockdown of PRDM16 during chondrogenic differentiation. 2) Identify PRDM16 DNA binding sites and histone modification pattern within PRDM16-enriched regions in chondrogenesis using chromatin immunoprecipitation-sequencing (ChIP-Seq). With the results and insights obtained from the K99 phase, we will then be able to 3) Unravel the function of PRDM16 in embryonic limb development and postnatal cartilage homeostasis in vivo using cartilage- specific conditional knock-out mouse models in R00 phase. This work will extend our understanding of the epigenetic regulation of chondrogenesis and limb development, and could provide important insights into the homeostasis of cartilage, as well as the refinement of new strategies for cartilage repair and regeneration.